Characterization of Pseudomonas aeruginosa Enoyl-Acyl carrier protein reductase (FabI):: a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis
Characterization of Pseudomonas aeruginosa Enoyl-Acyl carrier protein reductase (FabI):: a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis
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DOI：
10.1128/jb.181.17.5489-5497.1999
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发表时间：
1999-09-01
影响因子：
3.2
通讯作者：
Schweizer, HP
中科院分区：
文献类型：
--
作者：
Hoang, TT;Schweizer, HP
对铜绿假单胞菌编码烯酰酰基载体蛋白（ACP）还原酶的结构基因进行了克隆和测序。核苷酸序列分析表明，fabI可能是转录单元中包含编码未知功能ABC转运体atp结合蛋白的基因的最后一个基因，fabI蛋白的大小和初级序列与其他细菌烯酰acp还原酶相似，并且它分别包含fad依赖的吡啶核苷酸还原酶和葡萄糖/核糖醇脱氢酶家族的特征基序。染色体fabI基因被破坏，产生的突变体是有活力的，但仅具有野生型细胞提取物中烯基- acp还原酶活性的62%。fabi编码的烯酰acp还原酶活性依赖于NADH，并被三氯生抑制；fabI突变体的残留活性也依赖于NADH，但不受三氯生的抑制。纯化并鉴定了一种多组氨酸标记的FabI蛋白。纯化的FabI (i)可以使用NADH作为辅助因子，但不能使用NADPH；（ii）同时使用crotonyl辅酶A和crotonyl-ACP作为底物，虽然crotonyl-ACP的活性要高6倍；（iii）被低浓度的三氯生有效抑制，通过定点诱变产生A FabI Gly（95）到val活性位点的氨基酸取代，并纯化突变蛋白。突变体FabI蛋白保留了正常的烯酰acp还原酶活性，但具有高度的三氯生抗性。纯化的P. aeruginosa n -butyryl- homserine lactone （C-4-HSL）合成酶RhlI与FabI偶联后，可由crotonyl-ACP和s -腺苷蛋氨酸合成C-4-HSL。该反应依赖于NADH，并被三氯生抑制。在fabI突变体中，C-4-HSL和N-（3-氧）-十二烷基l -高丝氨酸内酯的水平降低了50%，证实了fabI在体内酰基化高丝氨酸内酯合成中的作用。
The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function, The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal fabI gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The fabI-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the fabI mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged FabI protein was purified and characterized. Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan, A FabI Gly(95)-to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified. The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant. When coupled to FabI, purified P. aeruginosa N-butyryl-L-homoserine lactone (C-4-HSL) synthase, RhlI, could synthesize C-4-HSL from crotonyl-ACP and S-adenosylmethionine. This reaction was NADH dependent and inhibited by triclosan. The levels of C-4-HSL and N-(3-oxo)-dodecanoyl L-homoserine lactones were reduced 50% in a fabI mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo.